Disruption of centrosome structure,chromosome segregation,and cytokinesis by misexpression of human Cdc14A phosphatase

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and gamma-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.     

Cdc15 is required for spore morphogenesis independently of Cdc14 in Saccharomyces cerevisiae

In Saccharomyces cerevisiae exit from mitosis requires the Cdc14 phosphatase to reverse CDK-mediated phosphorylation. Cdc14 is released from the nucleolus by the Cdc14 early anaphase release (FEAR) and mitotic exit network (MEN) pathways. In meiosis, the FEAR pathway is essential for exit from anaphase I. The MEN component Cdc15 is required for the formation of mature spores. To analyze the role of Cdc15 during sporulation, a conditional mutant in which CDC15 expression was controlled by the CLB2 promoter was used. Cdc15-depleted cells proceeded normally through the meiotic divisions but were unable to properly disassemble meiosis II spindles. The morphology of the prospore membrane was aberrant and failed to capture the nuclear lobes. Cdc15 was not required for Cdc14 release from the nucleoli, but it was essential to maintain Cdc14 released and for its nucleo-cytoplasmic transport. However, cells carrying a CDC14 allele with defects in nuclear export (Cdc14-DeltaNES) were able to disassemble the spindle and to complete spore formation, suggesting that the Cdc14 nuclear export defect was not the cause of the phenotypes observed in cdc15 mutants.     

Crm1-Mediated Nuclear Export of Cdc14 is Required for the Completion of Cytokinesis in Budding Yeast

The mitotic exit network (MEN) controls the exit from mitosis in budding yeast. The proline-directed phosphatase, Cdc14p, is a key component of MEN and promotes mitotic exit by activating the degradation of Clb2p and by reversing Cdk-mediated mitotic phosphorylation. Cdc14p is sequestered in the nucleolus during much of the cell cycle and is released in anaphase from the nucleolus to the nucleoplasm and cytoplasm to perform its functions. Release of Cdc14p from the nucleolus during anaphase is well understood. In contrast, less is known about the mechanism by which Cdc14p is released from the nucleus to the cytoplasm. Here we show that Cdc14p contains a leucine-rich nuclear export signal (NES) that interacts with Crm1p physically. Mutations in the NES of Cdc14p allow Clb2p degradation and mitotic exit, but cause abnormal morphology and cytokinesis defects at non-permissive temperatures. Cdc14p localizes to the bud neck, among other cytoplasmic structures, following its release from the nucleolus in late anaphase. This bud neck localization of Cdc14p is disrupted by mutations in its NES and by the leptomycin B-mediated inhibition of Crm1p. Our results suggest a requirement for Crm1p-dependent nuclear export of Cdc14p in coordinating mitotic exit and cytokinesis in budding yeast.     

Cdc14B depletion leads to centriole amplification, and its overexpression prevents unscheduled centriole duplication

Centrosome duplication is tightly controlled in coordination with DNA replication. The molecular mechanism of centrosome duplication remains unclear. Previous studies found that a fraction of human proline-directed phosphatase Cdc14B associates with centrosomes. However, Cdc14B's involvement in centrosome cycle control has never been explored. Here, we show that depletion of Cdc14B by RNA interference leads to centriole amplification in both HeLa and normal human fibroblast BJ and MRC-5 cells. Induction of Cdc14B expression through a regulatable promoter significantly attenuates centriole amplification in prolonged S phase-arrested cells and proteasome inhibitor Z-L(3)VS-treated cells. This inhibitory function requires centriole-associated Cdc14B catalytic activity. Together, these results suggest a potential function for Cdc14B phosphatase in maintaining the fidelity of centrosome duplication cycle.     

Human Cdc14A Reverses CDK1 Phosphorylation of Cdc25A on Serines 115 and 320

Human Cdc14A is an evolutionary conserved dual-specificity protein phosphatase that reverses the modifications effected by cyclin-dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates. Here, we show that human Cdc14A can dephosphorylate Cdc25A in vitro. Specifically, the Cdk1/Cyclin-B1-dependent phosphate groups on Ser115 and Ser320 of Cdc25A were found to be removed by Cdc14A. Cdc25A is an important cell cycle-regulatory protein involved in several cell cycle transitions and checkpoint responses and whose function and own regulation depend on complex phosphorylation/dephosphorylation-mediated processes. Importantly, we also show that the upregulation of Cdc14A phosphatase affects Cdc25A protein levels in human cells. Our results suggest that Cdc14A may be involved in the cell cycle regulation of Cdc25A stability.     

Dependence of Chs2 ER export on dephosphorylation by cytoplasmic Cdc14 ensures that septum formation follows mitosis

Cytokinesis, which leads to the physical separation of two dividing cells, is normally restrained until after nuclear division. In Saccharomyces cerevisiae, chitin synthase 2 (Chs2), which lays down the primary septum at the mother-daughter neck, also ensures proper actomyosin ring constriction during cytokinesis. During the metaphase-to-anaphase transition, phosphorylation of Chs2 by the mitotic cyclin-dependent kinase (Cdk1) retains Chs2 at the endoplasmic reticulum (ER), thereby preventing its translocation to the neck. Upon Cdk1 inactivation at the end of mitosis, Chs2 is exported from the ER and targeted to the neck. The mechanism for triggering Chs2 ER export thus far is unknown. We show here that Chs2 ER export requires the direct reversal of the inhibitory Cdk1 phosphorylation sites by Cdc14 phosphatase, the ultimate effector of the mitotic exit network (MEN). We further show that only Cdc14 liberated by the MEN after completion of chromosome segregation, and not Cdc14 released in early anaphase by the Cdc fourteen early anaphase release pathway, triggers Chs2 ER exit. Presumably, the reduced Cdk1 activity in late mitosis further favors dephosphorylation of Chs2 by Cdc14. Thus, by requiring declining Cdk1 activity and Cdc14 nuclear release for Chs2 ER export, cells ensure that septum formation is contingent upon chromosome separation and exit from mitosis.     

Visualization of a highly organized intranuclear network of filaments in living mammalian cells

For 30 years, the mammalian cell nucleus has been hypothesized to contain a filamentous framework, the nuclear matrix or karyoskeleton, which regulates nuclear structure and function. However, such an organized network of filaments has never been observed in living cells. Here we show that human Cdc14B phosphatase in living cells tightly associates with long filaments that begin at the nucleolar periphery and extend to the nuclear envelope, frequently making close connections with nuclear pore complexes. We demonstrate that Cdc14B contains a bipartite signal that directs it to the intranuclear filaments, and we also detect a small amount of Cdc14B on interphase and mitotic centrosomes. Furthermore, we show that Cdc14B is critical for the maintenance of proper nuclear structure together with polo-like kinase Plk1. This work provides the first direct evidence for the existence of an intranuclear filamentous framework in living mammalian cells and implicates Cdc14B in the control of mammalian nuclear architecture.     

Functional homology among human and fission yeast Cdc14 phosphatases

Budding and fission yeast Cdc14 homologues, a conserved family of serine-threonine phosphatases, play a role in the inactivation of mitotic cyclin-dependent kinases (CDKs) by molecularly distinct mechanisms. Saccharomyces cerevisiae Cdc14 protein phosphatase inactivates CDKs by promoting mitotic cyclin degradation and the accumulation of a CDK inhibitor to allow budding yeast cells to exit from mitosis. Schizosaccharomyces pombe Flp1 phosphatase down-regulates CDK/cyclin activity, controlling the degradation of the Cdc25 tyrosine phosphatase for fission yeast cells to undergo cytokinesis. In the present work, we show that human Cdc14 homologues (hCdc14A and hCdc14B) rescued flp1-deficient fission yeast strains, indicating functional homology. We also show that hCdc14A and B interacted in vivo with S. pombe Cdc25 and that hCdc14A dephosphorylated this mitotic inducer both in vitro and in vivo. Our results support a Cdc14 conserved inhibitory mechanism acting on S. pombe Cdc25 protein and suggest that human cells may regulate Cdc25 in a similar manner to inactivate Cdk1-mitotic cyclin complexes.     

The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B’s subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14BΔ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.     

Maintenance of G2 arrest in the Xenopus oocyte: a role for 14-3-3-mediated inhibition of Cdc25 nuclear import

Cdc2-cyclin B1 in the G2-arrested Xenopus oocyte is held inactive by phosphorylation of Cdc2 at two negative regulatory sites, Thr14 and Tyr15. Upon treatment with progesterone, these sites are dephosphorylated by the dual specificity phosphatase, Cdc25, leading to Cdc2-cyclin B1 activation. Whereas maintenance of the G2 arrest depends upon preventing Cdc25-induced Cdc2 dephosphorylation, the mechanisms responsible for keeping Cdc25 in check in these cells have not yet been described. Here we report that Cdc25 in the G2-arrested oocyte is bound to 14-3-3 proteins and that progesterone treatment abrogates this binding. We demonstrate that Cdc25, apparently statically localized in the cytoplasm, is actually capable of shuttling in and out of the oocyte nucleus. Binding of 14-3-3 protein markedly reduces the nuclear import rate of Cdc25, allowing nuclear export mediated by a nuclear export sequence present in the N-terminus of Cdc25 to predominate. If 14-3-3 binding to Cdc25 is prevented while nuclear export is inhibited, the coordinate nuclear accumulation of Cdc25 and Cdc2-cyclin B1 facilitates their mutual activation, thereby promoting oocyte maturation.     

Downregulation of PP2A(Cdc55) phosphatase by separase initiates mitotic exit in budding yeast

After anaphase, the high mitotic cyclin-dependent kinase (Cdk) activity is downregulated to promote exit from mitosis. To this end, in the budding yeast S. cerevisiae, the Cdk counteracting phosphatase Cdc14 is activated. In metaphase, Cdc14 is kept inactive in the nucleolus by its inhibitor Net1. During anaphase, Cdk- and Polo-dependent phosphorylation of Net1 is thought to release active Cdc14. How Net1 is phosphorylated specifically in anaphase, when mitotic kinase activity starts to decline, has remained unexplained. Here, we show that PP2A(Cdc55) phosphatase keeps Net1 underphosphorylated in metaphase. The sister chromatid-separating protease separase, activated at anaphase onset, interacts with and downregulates PP2A(Cdc55), thereby facilitating Cdk-dependent Net1 phosphorylation. PP2A(Cdc55) downregulation also promotes phosphorylation of Bfa1, contributing to activation of the "mitotic exit network" that sustains Cdc14 as Cdk activity declines. These findings allow us to present a new quantitative model for mitotic exit in budding yeast.     

Distinct nuclear and cytoplasmic functions of the S. pombe Cdc14-like phosphatase Clp1p/Flp1p and a role for nuclear shuttling in its regulation

Cdc14-like phosphatases regulate a variety of cell cycle events by dephosphorylating CDK sites. Their cell cycle-dependent changes in localization may be important to carry out distinct functions. Work in budding and fission yeast suggested that Cdc14-like phosphatases are inhibited by nucleolar sequestration. In S. cerevisiae, Cdc14p is released from the nucleolus by the FEAR network and Cdk1, whereas the S. pombe CDC14-like phosphatase Clp1p (also known as Flp1p) is released at mitotic entry by an unknown mechanism. The mitotic exit network (MEN) in S. cerevisiae and its homologous network, the septation initiation network (SIN), in S. pombe act through an unknown mechanism to keep the phosphatase out of the nucleolus in late mitosis. SIN-dependent cytoplasmic maintenance of Clp1p is thought to be essential for the cytokinesis checkpoint, which blocks further rounds of nuclear division until cytokinesis is completed. By targeting Clp1p to the nucleus or the cytoplasm, we demonstrate distinct functions for these pools of Clp1p in chromosome segregation and cytokinesis, respectively. Our results further suggest that the SIN does not keep Clp1p out of the nucleolus by regulating nucleolar affinity, as proposed for S. cerevisiae Cdc14p, but instead, Clp1p may be regulated by nuclear import/export.     

Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import

The Cdc25 phosphatases play crucial roles in cell cycle progression by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Cdc25A is an important regulator of the G1/S transition but functions also in the mitotic phase of the human cell cycle. In this paper, we investigate the sub-cellular localisation of exogenously expressed Cdc25A. We show that YFP-Cdc25A is localised both in the nucleus and the cytoplasm of HeLa cells and untransformed fibroblasts. Cell fusion assays and fluorescence loss in photobleaching (FLIP) assays reveal that the localisation is dynamic and the protein shuttles between the nucleus and the cytoplasm. We demonstrate that nuclear export of Cdc25A is partly mediated by an N-terminal nuclear export sequence (NES), in a manner not sensitive to the Exportin 1-inhibitor leptomycin B. A nuclear localisation signal (NLS) is also characterised, mutation of which leads to cytoplasmic localisation of Cdc25A. Our results imply that the Cdc25A phosphatase may interact with substrates and regulators both in the nucleus and the cytoplasm.     

Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25C(S214A) into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.     

Cdc14A and Cdc14B Redundantly Regulate DNA Double-Strand Break Repair

Cdc14 is a phosphatase that controls mitotic exit and cytokinesis in budding yeast. In mammals, the two Cdc14 homologues, Cdc14A and Cdc14B, have been proposed to regulate DNA damage repair, whereas the mitotic exit and cytokinesis rely on another phosphatase, PP2A-B55α. It is unclear if the two Cdc14s work redundantly in DNA repair and which repair pathways they participate in. More importantly, their target(s) in DNA repair remains elusive. Here we report that Cdc14B knockout (Cdc14B^−/−) mouse embryonic fibroblasts (MEFs) showed defects in repairing ionizing radiation (IR)-induced DNA double-strand breaks (DSBs), which occurred only at late passages when Cdc14A levels were low. This repair defect could occur at early passages if Cdc14A levels were also compromised. These results indicate redundancy between Cdc14B and Cdc14A in DSB repair. Further, we found that Cdc14B deficiency impaired both homologous recombination (HR) and nonhomologous end joining (NHEJ), the two major DSB repair pathways. We also provide evidence that Cdh1 is a downstream target of Cdc14B in DSB repair.     

Greatwall maintains mitosis through regulation of PP2A

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B–Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B–Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B–Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B–Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A‐dependent dephosphorylation of cyclin B–Cdc2 substrates is increased in GW‐depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B–Cdc2 but also by the regulation of PP2A by GW.     

Human Cdc14A phosphatase modulates the G2/M transition through Cdc25A and Cdc25B

The Cdc14 family of serine-threonine phosphatases antagonizes CDK activity by reversing CDK-dependent phosphorylation events. It is well established that the yeast members of this family bring about the M/G1 transition. Budding yeast Cdc14 is essential for CDK inactivation at the end of mitosis and fission yeast Cdc14 homologue Flp1/Clp1 down-regulates Cdc25 to ensure the inactivation of mitotic CDK complexes to trigger cell division. However, the functions of human Cdc14 homologues remain poorly understood. Here we have tested the hypothesis that Cdc14A might regulate Cdc25 mitotic inducers in human cells. We found that increasing levels of Cdc14A delay entry into mitosis by inhibiting Cdk1-cyclin B1 activity. By contrast, lowering the levels of Cdc14A accelerates mitotic entry. Biochemical analyses revealed that Cdc14A acts through key Cdk1-cyclin B1 regulators. We observed that Cdc14A directly bound to and dephosphorylated Cdc25B, inhibiting its catalytic activity. Cdc14A also regulated the activity of Cdc25A at the G2/M transition. Our results indicate that Cdc14A phosphatase prevents premature activation of Cdk1 regulating Cdc25A and Cdc25B at the entry into mitosis.     

Chk1-Dependent Regulation of Cdc25B Functions to Coordinate Mitotic Events

The coordination of mitotic spindle formation and chromatin condensation is an essential prerequisite for successful mitosis. Both events are thought to be initiated by cyclin B/Cdk1, whose initial activation occurs in late prophase at the centrosomes. Recently, we have shown that Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B/Cdk1. Here, we demonstrate that inhibition of Chk1 kinase induces mitotic entry with regular spindle assembly but aberrant and mislocalized chromatin. This effect, which we have termed the ‘paraspindle’ phenotype, was reverted by downregulation of Cdc25B phosphatase using siRNA, which restored normal mitosis with regular chromatin. Analogous to Chk1 inhibition, the ‘paraspindle’ phenotype was induced by overexpression of Cdc25B but not Cdc25A. Our results suggest that Chk1 functions to coordinate mitotic events through regulation of Cdc25B.     

Novel role for Cdc14 sequestration: Cdc14 dephosphorylates factors that promote DNA replication

The phosphatase Cdc14 is required for mitotic exit in budding yeast. Cdc14 promotes Cdk1 inactivation by targeting proteins that, when dephosphorylated, trigger degradation of mitotic cyclins and accumulation of the Cdk1 inhibitor, Sic1. Cdc14 is sequestered in the nucleolus during most of the cell cycle but is released into the nucleus and cytoplasm during anaphase. When Cdc14 is not properly sequestered in the nucleolus, expression of the S-phase cyclin Clb5 is required for viability, suggesting that the antagonizing activity of Clb5-dependent Cdk1 specifically is necessary when Cdc14 is delocalized. We show that delocalization of Cdc14 combined with loss of Clb5 causes defects in DNA replication. When Cdc14 is not sequestered, it efficiently dephosphorylates a subset of Cdk1 substrates including the replication factors, Sld2 and Dpb2. Mutations causing Cdc14 mislocalization interact genetically with mutations affecting the function of DNA polymerase epsilon and the S-phase checkpoint protein Mec1. Our findings suggest that Cdc14 is retained in the nucleolus to support a favorable kinase/phosphatase balance while cells are replicating their DNA, in addition to the established role of Cdc14 sequestration in coordinating nuclear segregation with mitotic exit.